Most of the chemical compounds present in living organisms contain skeletons of covalently bonded carbon atoms (C-C-C-C). These compounds are known as organic compounds, because most of these are either present in, or produced by living things. Organic compounds are the major components of cells and tissues. They provide energy for life processes, participate in and regulate metabolic reactions, and transmit information. Organic macromolecules in living organisms can be classified as either carbohydrates, proteins, lipids, or nucleic acids, among others. These macromolecules are always made of smaller subunits. The subunits of macromolecules are held together with covalent bonds, and have different structures and properties. For example, lipids made of fatty acids have many C-H bonds and relatively little oxygen, while proteins made of amino acids have amino groups (-NH2) and carboxyl (-COOH) groups. These characteristic groups impart different chemical properties to macromolecules--for example, monosaccharides such as glucose are polar and soluble in water, while lipids are nonpolar and insoluble in water.
Carbohydrates are compounds that contain carbon, hydrogen and oxygen. Carbohydrates include a variety of compounds, such as sugars, starches, and celluloses. While sugars and starches serve as energy sources for cells; celluloses are structural components of the walls that surround plant cells. The term carbohydrate literally means "hydrated (H20) carbon" Carbohydrates may contain one sugar molecule (monosaccharides), two sugar molecules (disaccharides), or many sugar units (polysaccharides). In this lab, we will be concerned with the nature and activities of the carbohydrates and with their structure. Note: structure dictates how the carbohydrate will react under certain conditions.
Since carbohydrates are readily identified by color change in specific reactions, we will explore some of these methods of identification as we carry out specific tests for particular carbohydrates. Solutions of the following mono-, di-, and polysaccharides are available: (a) glucose, (b) fructose, (c) galactose, (d) xylose, (e) lactose, (f) maltose, (g) sucrose, and (h) starch. These solutions are available at both 1% and 6% concentrations (percent solutions are by weight/volume, thus 1% is 1 gram in 100 ml of solvent).
A ninth unknown carbohydrate
will also be run through the tests with the known sugars. Your challenge
is to identify the unknownís chemical features and suggest a possible structure
as best you can by its test results. This unknown may or may not be one
of the carbohydrates listed above.
TESTS FOR REDUCING SUGARS (Multiple Fume Hoods required)
Reducing sugars are those that have a free or potentially free aldehyde or ketone. In a solution of pH 8 or higher the sugar is capable of reducing certain weak oxidizing agents such as cupric hydroxide along with a resultant oxidation of the carbonyl group of the sugar. Both Benedicts and Barfoeds tests identify reducing sugars. The following reaction is an example:
glucose + 2Cu(OH)2 --------- > Cu2O + 2H2O + glucose
(oxidized cupric ions: blue) (reduced cuprous ions: red)
1. Place 3 ml of Benedict's reagent in each of eight labeled test tubes and add 300µl drops of 1% solutions of each of the known carbohydrates to be tested. Shake each test tube to assure thorough mixing.
2. Place the tubes in a boiling water bath for 3 minutes. At the end of that time, allow the tubes to cool and record your results on the chart appended.
Barfoed's test. This test can distinguish monosaccharides from di- and polysaccharides because with the conditions of lower pH and shorter incubation time, only monosaccharides can react fast enough to reduce copper ions. The reagent is similar to Benedict's except that the pH is lower (around pH 4.5) and heating time is reduced to 2 minutes. Do not heat longer than 2 minutes. Longer heating may cause hydrolysis of the glycosidic linkage, thus breaking disaccharides to monosaccharides.
1. Put 3ml of Barfoed's solution in each of eight labeled test tubes and add to each tube 500 µl drops of the 1% sugar solution to be tested.
2. Place the tubes in a boiling water bath and observe at 1 minute but continue to heat until 2 minutes. Record your results after 2 minutes. A rusty or brownish-red color will indicate monosaccharides, no color change or weak change will indicate di and polysaccharides.
Mineral acids (HCl and H2SO4) react with carbohydrates to form furfural, hydroxymethyfurfural or levulinic acid by dehydration. Pentoses produce furfural acid, while hexoses produce hydroxymethyfurfural. The hydroxymethyfurfural further reacts with the acid to form levulinic acid and formic acid. In these tests, polysaccharides and disaccharides are hydrolyzed, which breaks the glycoside linkages to yield monosaccarides. This allows them to react as monosaccharides. Several tests are based on these reactions. We will examine three of them.
Selivanoff's Test. This test is used to differentiate between ketoses and aldoses. The reagent is a solution of resorcinol in concentrated HC1. The acid when heated along with a sugar will produce furfural or hydroxymethylfurfural, which further reacts to give a red color. Ketoses react more quickly than aldoses and thus the reaction time is a means of separation or detection. Ketoses react within 1 minute of heating while aldoses will require several minutes. Disaccharides containing fructose should react intermediately between that of fructose alone and one of the aldoses.
1. Place 350 µl drops of the 1% sugar solution in each test tube. Add 3ml of Selivanoffís reagent to each tube.
2. Place the tubes in the hot water bath, time the reactions and begin your observations immediately. Record your results in Table 1 as before.
Bial's Test. This test is used to detect the presence of furanoses (five-membered rings). These sugars, or compounds containing them, react with Bial's reagent to give a green or olive colored solution. Furan rings contain five carbons but sugars with furan rings can contain more carbons outside the ring, and all sugars with a furan ring will react in Bial's test. The pentose furanoses will react with Bial's reagent to form green solution, as the hexose furanose will react to form olive/brown solution. Bial's contains orcinol (5-methyl- 1,3 dihydroxybenzene), the parent compound of the litmus dyes in concentrated HCl.
1. Place 35 µl drop of the 1% sugar solution in each test tube. Add 3ml of Bial's reagent to each test tube.
2. Place each tube in the hot water bath for 5 minutes. At the end of that time take the test tubes out of the hot water bath and record your observations.
Mucic Acid Test. This test is one in which concentrated HNO3 is heated along with an aldose sugar to give a dicarboxylic acid. Nitric acid is able to oxidize the terminal groups of aldoses, but leaves the secondary hydroxyl groups unchanged. The dicarboxylic acid formed from galactose is called mucic acid and is insoluble in cold aqueous solution. Those acids formed from the other common sugars are soluble in H20. Thus the formation of the insoluble precipitate is an indication of the presence of galactose. This test requires several hours to complete and consequently the TAs will do it for you and you can mark the results in the proper place in Table 1. The procedure we will follow will be to place 1ml of 6% carbohydrate solution in each of the test tubes. We will then add 1ml of concentrated HNO3 and heat in a boiling bath for 1 1/2 hours. We will then remove the tubes, let them stand overnight and read the subsequent results.
Iodine (iodine-potassium iodide, I2KI) staining distinguishes starch (a polysaccharide) from monosaccharides, disaccharides, and other polysaccharides. The basis for this test is that starch is a coiled polymer of glucose. Iodine interacts with these coiled molecules and becomes bluish black. Other non-coiled carbohydrates do not react with iodine. Therefore, a bluish black color is a positive test for starch, and a yellow-ish brown color (i.e., no color change) is a negative test for starch. Glycogen, the common polysaccharide in animals, has a slight difference in structure and produces only an intermediate color reaction. Test each of the known sugars for the presence of starch.
1. Place 35 µl drops of the 1% sugar solution in each test tube. Add 35 µl drop of IKI reagent to each tube.
2. Keep the tubes at room temperature. Record your results in Table 1.
2. Barfoed's - test for reducing sugars that are monosaccharides. Similar to Benedict's but lower pH and shorter time. Only monosaccharides will reduce copper and change color due to formation of orange/red precipitate.
3. Selivanoff's - test for ketose vs aldose. CAUTION: ACID - Color turns red fastest if the sugar is a ketose. Red < 1 min. (fastest) is a monosaccharide ketose, Red ~ 1 min. (slightly slower) is a disaccharide ketose, Red > 1 min. (longest) is aldose.
4. Bial's - tests for furanose ring. CAUTION: ACID - Yellow color turns greenish if sugar is a furanose (has a five membered ring like furan). If sugar is a pentose-furanose: color turns green/olive; a hexose (or higher)-furanose: color turns a muddy brown, or if the sugar is a pyranose: no change in color.
5. Mucic Acid - DEMO test for galactose. CAUTION: ACID - aldose + acid forms dicarboxylic acid. Mucic acid is insoluble in H20 and forms white precipitate.
6. Iodine - test for the presence of starch. If starch is present, the addition of IKI will turn the substance being tested to a blue-black color.
1. Obtain an unknown solution from your laboratory instructor. Record its number in a Table.
2. Obtain 6 clean test-tubes.
3. Perform each of the carbohydrate diagnostic tests on the unknown.
4. Record your results in Table 1 and describe the chemical characteristics of your unknown. What do the results tell you about the chemical characteristics of your unknown? If any of your tests are inconclusive, repeat those tests so you are certain of the data for your unknown.